In Drosophila, two groups of genes, the Polycomb group (PcG) and the Trithorax group (TrxG) are important for inheritance of the silenced and the active chromatin state, respectively. Regulatory elements called Polycomb group response elements (PREs) are cis-acting DNA sequences required for the recruitment of chromatin-modifying PcG protein complexes. TrxG proteins act through either the same or overlapping cis-acting sequences as PcG proteins. Our group is working on understanding how PcG and TrxG proteins get recruited to the DNA. Polycomb group response elements (PREs) are DNA elements through which the Polycomb group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated in two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many different proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181-bp PRE from the Drosophila engrailed gene. Our goal is to define all of the DNA binding proteins required for the activity of this PRE, and to ask whether all PREs are made up of binding sites for the same proteins. Disruption of any of seven different binding sites within this 181-bp PRE disrupts PRE function. The proteins that bind five of these sites are known: Pho/Phol, GAF/Psq, Zeste, Dsp1, and members of the Sp1/KLF family. Our lab identified Pho and Phol and have been studying their function. Pho and Phol are related proteins that bind the same sequence and have some redundant activities. Pho and Phol are required for PcG protein function and for binding of PcG proteins to the DNA. pho, phol double mutant animals have a PcG phenotype clearly indicating they play a central important role in PcG repression. More recently we have been studying the Sp1/KLF family proteins in Drosophila, to try to determine whether a family member plays a central role in PcG protein silencing. This family of proteins encodes transcription factors and has been extensively studied in mammals. There are 20 Sp1/KLF family members in mammals. In Drosophila there are 9 Sp1/KLF family members, and eight of them bind to a site in the 181-bp PRE we study. We derived a consensus-binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila. We identified a Sp1/KLF family member, Spps, important for PRE activity. Spps encodes a Sp1/KLF family member most highly related to the ubiquitous mammalian transcription factor Sp3. Spps is a ubiquitous protein, expressed at all stages of development. Strikingly, Spps co-localizes with the PcG protein Psc on polytene chromosomes. The co-localization of Spps with Psc is even greater than the co-localization of Pho with Psc. Further, we showed by chromatin immunoprecipitation that Spps binds to both the en and the bxd PREs in S2 cells and larvae. Our recent genome-wide studies confirm the presence of Spps at PREs. We generated a deletion of the Spps transcription unit by homologous recombination and were surprised that Spps mutants live until the pharate adult stage with no obvious PcG phenotype. However Spps mutants suppress PRE-mediated pairing-sensitive silencing, suggesting that Spps does act in PcG repression. Finally, Spps mutations enhance the Pho phenotype, suggesting these two proteins act together to recruit PcG proteins to the DNA. We have recently identified another DNA binding protein associated with PREs. Current work aims to identify the role of these various proteins in PcG protein recruitment. Another important aspect of our work is to ask whether all PREs are alike. In this regard we characterized PREs from the Drosophila invected gene as well as an additional PRE from the engrailed gene. Our work suggests that there is not one way to make a PRE. For example, it was suggested in the literature that two Pho/Phol binding sites are required for PRE function. Our laboratory found that this is true for some PREs, but in the 181-bp PRE, only one Pho/Phol binding site is necessary for PRE function. Our data show that the number, spacing, order of binding sites vary in different PREs and these differences can have functional consequences. Our current model is that PREs are a group of related elements that can diverge rapidly and vary in activity depending on the target gene they regulate. The PRE we study is from the Drosophila engrailed gene. engrailed encodes a homeodomain protein that plays an important role in the development of many different parts of the embryo including the formation of the segments, nervous system, head, and gut. It also plays a very important role in the development of the adult, specifying the posterior compartment of each imaginal disc. Accordingly, engrailed is expressed in a very specific and complex manner in the developing organism. The 181-bp engrailed PRE we have been studying is located near the engrailed promoter from 576 to 395bp upstream of the transcription start site. We are interested in determining the role of this PRE in the control of engrailed expression. One of the first things we learned in our studies is that this PRE is redundant with other flanking PREs in the endogenous engrailed gene. There is another strong PRE located from -1100 to -1500 bp and probably other weak PREs nearby. In fact, when we examined the location of Ph and Pho proteins on engrailed DNA by chromatin immunoprecipation (ChIP), we found that they are bound to a 2.5 kb region extending from the engrailed promoter to about 2.5kb upstream. Therefore, it is perhaps not too surprising that a 500bp deletion that includes the 181-bp PRE and flanking sequence did not lead to ectopic engrailed expression. The remaining PREs were apparently sufficient to recruit PcG proteins. However, what was surprising to us was that loss of this DNA lead to a loss of function phenotype, suggesting that this DNA must also play a positive role in the expression of engrailed. Our experiments suggest that there are multiple positive elements either overlapping with or coincident with the PREs. Our work also suggests that PREs can mediate looping, either with distant PREs or with distant enhancers. Thus; PREs can either activate or repress transcription, dependent on the context. This is an important result consistent with the idea that PREs are also the site of action of Trithorax group proteins. Finally, we have also found that sequences required for promoter-enhancer communication (promoter tethering elements) are located within or near the engrailed PREs. The regulatory sequences for the engrailed gene extend over a 70 kb region. Our lab used reporter constructs to find sequences important for expression in stripes, the nervous system, the head, etc. There are discrete regulatory elements located throughout the 70kb region for most of engrailed's expresion patterns. However, we were not able to locate the enhancer responsible for expression in the imaginal discs, the precursors of the adult structure, in a small DNA fragment. Our data suggests that a complex interplay of regulatory elements is necessary for expression in imaginal disks. Our data suggests that the chromatin structure plays an important role in regulating expression from the imaginal disc enhancer. These results challenge the idea that all enhancers are discrete elements that operate in a context-independent way, suggesting that some enhancer activity is highly dependent on chromatin context.